Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1

Background N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. Results We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved –GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. Conclusion Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03225-4.

In this study, we characterized two amidases from Deinococcus indicus DR1, namely Ami1 Di and Ami2 Di .D. indicus DR1 belongs to the Deinococcaceae family and is a rod-shaped, red-pigmented bacterium majorly known for high arsenic tolerance [35].Our results indicate that both Ami1 Di and Ami2 Di from D. indicus are able to suppress cell separation defects in E. coli amidase mutants.Computational modeling revealed that both proteins possess the Amidase_3 catalytic domain with conserved -GHGG-motif and Zn 2+ -binding sites.Structures of both Ami1 Di and Ami2 Di were predicted by AlphaFold2.Structural similarity revealed Ami1 Di being a closer homolog to AmiA of E. coli and may follow the same amidase/activator model.Our study is the first step in characterizing amidases from an extremophile D. indicus and can help uncover their role in maintaining complex cell envelopes.

Strains, media, and growth conditions
D. indicus DR-1 cells were grown in PYE (peptone yeast extract) [36] medium supplemented with 1 mM MgSO 4 and 1 mM CaCl 2 at 30 °C.E. coli DH5α cells were used for cloning, and BL21 (DE3) was used for protein induction and purification.For plasmid selection, E. coli cells were grown in LB (Luria Bertani) medium at 37 °C with kanamycin antibiotic (50 µg/mL).For induction of genes encoded under the control of L-arabinose or lactose inducible promoter, cells were grown in the presence of 0.4% L-arabinose or 0.5 µM IPTG (isopropyl-β-d-1thiogalactopyranoside).The strains and plasmids used are mentioned in Table 1.Antibiotics used in this study were purchased from Sigma Aldrich (Milwaukee, U.S.A), and media were purchased from Himedia (Mumbai, India).

Microscopy
Cells were collected at different time points and immobilized on 1% 1 X PBS agarose padded slides and were subjected to phase contrast microscopy using a Nikon Eclipse Ts2R microscope (Nikon, Japan) attached with a Nikon DS-Fi3 camera equipped with Nikon Plan Fluor 100X oil Ph3 objective.Time-lapse imaging of live cells harvested at mid-exponential phase (OD 600 ∼ 0.4) was performed on LB 0.7% agarose padded slides supplemented with or without 0.4% L-arabinose using a Nikon Eclipse Ti microscope (Nikon, Japan) with Nikon DS-U3 camera and Plan Apo 100 X oil objective.Image processing was performed with ImageJ [37] and Adobe Photoshop CS6 (Adobe Inc.U.S.A).

Complementation assay
D. indicus putative amidase ami1 Di and ami2 Di was cloned into the pBAD18 vector under the control of L-arabinose promoter and transformed into E. coli ∆amiAC (RP21) competent cells resulting in the formation of constructs E. coli ∆amiAC/pBADami1 Di (RP101) and E. coli∆amiAC/pBADami2 Di (RP104).To check for complementation, cells were serially diluted at a ratio of 1:100, induced with 0.4% L-arabinose at OD 600 ∼ 0.2, and subjected to phase contrast imaging.E.coli ∆amiAC/pBAD18 (empty vector) induced with 0.4% L-arabinose was used as a control.Cells were counted for the number of singlets, doublets, triplets, and chains using ImageJ [37].

Site-directed mutagenesis
Multiple sequence alignment using ClustalW [38] identified conserved active site residue between E. coli AmiC, C. crescentus AmiC, and D. indicus Ami1 sequences.Active site mutant was generated using the QuickChange Method (Stratagene, La Jolla, CA) [39].Point mutation at position 161 was created by replacing Histidine with Alanine.The parental plasmids were digested with DpnI, and mutant plasmids were used to transform E. coli DH5α with selection on LB + kanamycin plates.Active site mutants were further confirmed by Sanger sequencing.

Construction of the recombinant his-tag fusion proteins
The 1.17-kb gene encoding Ami1 D.i and 1.7-kb encoding Ami2 D.i were tagged with 6xHis sequence at the C-terminus using primers mentioned in supplementary Table 2. PCR purified fragments of Ami1 Di and Ami2 Di were digested with EcoRI and HindIII restriction enzymes and ligated within pET28b vector.After isolation, recombinant plasmids were confirmed by Sanger sequencing and introduced into E. coli BL21 (DE3) for protein production and purification.

Western blotting analysis
Purified proteins (Ami1 D.i and Ami2 D.i ) were separated based on their molecular weight on SDS-PAGE gel (12% w/v) and transferred onto Polyvinylidenedifluoride (PVDF) membrane.After blocking with 5% skim milk in 1 X PBS buffer, the membrane was probed with rabbit anti-His tag monoclonal antibody (1:3000 dilution, Invitrogen, Waltham, MA, USA) overnight at 4 °C.The membrane was washed 3 times with PBST and probed with horseradish peroxidase-conjugated anti-rabbit antibodies (1:10000 dilution, Invitrogen, Waltham, MA, USA).Final blot was developed with Bio-Rad Clarity and Clarity Max

PG purification and hydrolytic activity assay
Peptidoglycan from D. indicus DR-1 was purified as described previously with slight modifications [41].About 2 L of D. indicus was cultured in TSB medium and grown to an OD 600 ̴ 0.8.Cells were pelleted down at 12,000 g for 20 min and washed twice with distilled water (0.2 g/mL).The cell suspension was added dropwise into 8% boiling SDS with vigorous stirring.The solution was boiled for 2 h, and the lysate was allowed to cool down at room temperature overnight.Afterward, the solution was pelleted by ultracentrifugation at 120,000 g for 90 min at room temperature.The insoluble peptidoglycan obtained was washed at least eight times with distilled water to remove residual SDS (SDS concentration < 1 µg/mL).
The final concentration of SDS in insoluble peptidoglycan was determined by the Methylene Blue assay [42].
Isolated PG was resuspended in 3 mL of Tris (10 mM) buffer.Remazol brilliant blue (RBB) labeled sacculi was prepared as described previously [26].0.2 g of insoluble peptidoglycan was resuspended in 10 mL of 0.25 M NaOH containing 20 mM RBB.The suspension was incubated overnight at 37 °C and then washed repeatedly with distilled water until the supernatant was clear.For the hydrolytic activity assay, 100 µL RBB-labeled sacculi were incubated with about 2 µg of purified protein in 100 µL of PBS buffer (10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4) and incubated at 37 °C for different time points.Lysozyme (2 µg) and protein wash buffer were used as control.Reactions were pelleted down at 16,000 g for 10 min.Hydrolyzed PG was determined spectrophotometrically at 595 nm by measuring the concentration of released RBB dye in the collected supernatant.

Computational models of Ami1 Di and Ami2 Di
The sequences of Ami1 Di (Accession id: WP_229844239) and Ami2 Di (Accession id: WP_088250252) from the D. indicus DR1 genome were extracted from NCBI.We employed AlphaFold2 [43] as implemented in ColabFold [44] pipeline.We searched for suitable templates and carried out three rounds of AMBER-based energy relaxation, yielding a total of five structural models.These models were subsequently ranked based on their pLDDT scores ranging from 0 to 100.A higher pLDDT score is indicative of a more reliable and high-quality structure.
The structurally equivalent residues for the zinc (Zn 2+ ) binding site in both amidases were identified visually using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).We used AmiC from Escherichia coli (PDB id: 4BIN) as the reference protein to identify the structurally equivalent residues in Ami1 Di and Ami2 Di .Structurally similar amidase proteins from various organisms using Ami1 Di and Ami2 Di as query structures were searched using the DALI server [45] and structures having a Z-score above 10 were considered for further analysis to identify structural homology.

Phylogenetic tree analysis
We generated phylogenetic trees using the MEGA 11 [46] tool for both Ami1 Di and Ami2 Di by comparing them with structurally similar amidase proteins identified through the DALI server, as well as AmiC from C. crescentus.However, we conducted two separate analyses to build phylogenetic trees, one for Ami1 Di and one for Ami2 Di .The Ami1 Di analysis involved 16 amino acid sequences, consisting of 14 sequences having structural homology with Ami1 Di according to Z-score predicted by DALI server, one sequence representing Ami1 Di from D. indicus, and one sequence corresponding to AmiC from C. crescentus.Meanwhile, the Ami2 Di analysis comprised 14 amino acid sequences, including 12 sequences having structural homology to Ami2 Di from DALI, one sequence from D. indicus representing Ami2 Di , and one sequence representing AmiC from C. crescentus.We utilized the ClustalW algorithm to align the protein sequences.The evolutionary history was deduced utilizing the Neighbor-Joining method [47], with a bootstrap consensus tree based on 1000 replicates to represent the evolutionary relationships among the analyzed taxa [48].Branches representing partitions that appeared in less than 50% of the bootstrap replicates were collapsed.The percentage of replicate trees where the associated taxa clustered together during the bootstrap test (1000 replicates) is displayed adjacent to the branches.Evolutionary distances were calculated using the Jones-Taylor-Thomton (JTT) model, a matrix-based method [49], and are expressed in terms of the number of amino acid substitutions per site.Ambiguous positions were excluded for each sequence pair (pairwise deletion option).The final dataset consisted of 870 positions for Ami1 Di and 895 positions for Ami2 Di , and these positions indicate the total number of aligned sites in the amino acid sequence that were used to construct the phylogenetic tree and infer the evolutionary relationship.

Phylogenetic analysis
Blast analysis revealed that the D. indicus genome encodes two cell wall amidases annotated here as Ami1 Di (WP_229844239) and Ami2 Di (WP_088250252).Both proteins are predicted to have approximately 30 amino acid signal peptide sequences at the N-terminus.Unlike E. coli AmiC [50], the AMIN domain was absent in both D. indicus amidase proteins (Fig. 1A), and both proteins lack conventional cell wall targeting and peptidoglycan binding domains.At the C-terminus, both proteins show high similarity to the Amidase_3 domain, indicating that they belong to the Amidase_3 family, which includes zinc-dependent enzymes (Fig. 1A).The Amidase_3 domain of Ami2 Di was found to be smaller in size (176 aa) in comparison to the Amidase_3 domain of Ami1 Di .
Figure 1B shows a phylogenetic tree generated by the MEGA 11.0 program, where we found that both amidases are closely associated with the N-acetylmuromoyl-L-alanine amidase from two distinct organisms.Specifically, Ami1 Di demonstrated a close relationship with Peptoclostridium difficile (Fig. 1B-i), while Ami2 Di exhibited a close association with Paenibacillus polymyxa (Fig. 1Bii).These highlight possible reasons for their functional and structural differences.Using the DALI server, we predicted a total of 14 structural homologs for Ami1 Di and 12 structural homologs for Ami2 Di , all of which had a Z-score above 10, as documented in Table S1.Notably, Ami1 Di exhibited two distinct hits that were presented in Ami2 Di .These unique hits in Ami1 Di included the structure of the putative N-acetylmuromoyl-L-alanine amidase from Neisseria meningitidis and the Thermosome subunit from Methanococcoides burtonii.The remaining hits were common to Ami1 Di and Ami2 Di with varying Z-scores (Table S1).

Ami1 Di and Ami2 Di from D. Indicus are functional in E. Coli
To investigate the role of D. indicus amidases in daughter cell separation, we introduced full-length ami1 Di on pBAD18 plasmid into E. coli cells lacking both amiA and amiC (RP101).RP101 cells, when grown in the presence of L-arabinose, showed an increase in single and paired cells and a decrease in chains (Fig. 2A).In contrast, RP101 cells grown in LB medium supplemented with glucose and control cells (RP21) harboring empty pBAD vector, displayed 71% and 67% of cells attached in chains.Quantitative analysis of experiments revealed that due to the induction of Ami1 Di with L-arabinose, there was about a 40% decrease in chains, a 32% increase in singlets, a 30% increase in doublets, and a 5% increase in triplets (Fig. 2B) compared to the control cells carrying Fig. 2 Ami1 Di can suppress cell separation defects in E. coli amidase mutant.(A) Phase contrast micrographs representing cells of strain RP21 (MG1655 ΔamiA::frt ΔamiC::frt /pBAD18), RP101 (MG1655 ΔamiA::frt ΔamiC::frt /pBAD18ami1 Di ) induced with 0.4% L-arabinose (ara).Cells were grown in LB medium at 37 °C to an OD 600 ∼ 0.2 and induced with 0.4% L-arabinose (ara).RP101 strain grown with 0.2% glucose (glu) was used as a negative control.After 6 h of induction, cells were collected and 6 µL was immobilized on 1XPBS agarose pad and imaged.In the presence of 0.4% L-arabinose complementation of chain-forming double-amidase mutant by ami1 Di resulted in a reduction in the chaining phenotype.(B) Quantitative analysis of phase contrast micrographs of strains RP21 and RP101.Cells were counted as mentioned in Materials and Methods.RP101 shows a reduction in chains and an increment in singlets and doublets.Datasets are from three independent experiments, and error bars represent standard deviation.P value = RP21 vs. RP101 + 0.2% glu (glucose), p < 0.05**, RP21 vs. RP101 + 0.4% ara (L-arabinose), p < 0.05**.No. of cells -Total number of cells counted for each strain empty vector, suggesting that Ami1 Di can suppress cell separation defects in E. coli amidase mutants.
Similarly, we expressed Ami2 Di under the control of L-arabinose promoter in E. coli cells deleted for both AmiA and AmiC (RP104).Post induction with L-arabinose, a significant decrease in the number of cells in chains was observed (Fig. 3A), indicating that Ami2 Di may have amidase activity and can suppress cell separation defects.Compared to control cells grown in glucose, where 60% of cells were present in chains, in RP104 under L-arabinose induction only about 14% of the cell population was in chains (Fig. 3B).Increase in single cells (̴ 45%) and double cells (̴ 31%) was also observed suggesting that cell division and cytokinesis were taking place in RP104 (Fig. 3B).Taken together our results indicate that both Ami1 Di and Ami2 Di have cell wall hydrolytic activity and may play a role in daughter cell splitting after cell division.

Overexpression of ami_2 Di leads to cell lysis
Overexpression of N-acetylmuramyl-L-alanine amidases can lead to bacteriolysis, suggesting amidases to be powerful lytic enzymes [15,51].To examine if over activity of D. indicus amidases in E. coli would lead to cell lysis, RP101 and RP104 cells were induced with 0.4% L-arabinose.After 4 h post-induction with L-arabinose, cells overexpressing ami1 Di showed decreased growth (Fig. 4B) and cell lysis (Fig. 4A).However, there was a drastic reduction in cell viability (Fig. 4B) and enhanced lysis in cells overexpressing ami2 Di (Fig. 4A), suggesting that ami2 Di probably has stronger cell wall hydrolytic activity.In contrast, the control cell harboring empty pBAD plasmid did not exhibit cell lysis under similar conditions.

Structural analysis of Ami1 Di and Ami2 Di
AlphaFold2 predicted the structures of both Ami1 Di and Ami2 Di with reasonable accuracy, as indicated by their respective pLDDT scores of 82.7 and 79.1, along with corresponding ptm scores of 0.593 and 0.616 (Fig S1 &  S2).These scores demonstrate the suitability of these predicted structures for further analysis.Ami1 Di is predicted to possess a two-domain structure, consisting of a β-sandwich N-terminal domain and an α/β C-terminal domain connected by an alanine-rich linker region (Fig. 5A).Notably, the initial 25 residues of Ami1 Di are predicted to be disordered.Ami2 Di is also a two-domain protein but exhibits significant differences compared to Ami1 Di .It is predicted as a three-domain protein, featuring a β-sandwich N-terminal domain, an α/β C-terminal domain, and an additional β-sandwich domain situated between the two primary domains (Fig. 5D).Interestingly, this α/β domain is shared between both proteins.We hypothesize that it is functionally relevant as the Zn 2+ -binding region and -GHGG-motif is present in this domain.Remarkably, the α/β domain is shared among all structurally analogous amidase proteins exhibiting a Z-score above 10, as predicted by the DALI server.Specifically, Ami1 Di demonstrates a closer structural resemblance to the novel amidase from Mycobacterium tuberculosis (PDB id: 4lQ6), with a Root Mean Square Deviation (RMSD) of 2.89 Å calculated over 176 residues, compared to other amidases from various organisms.Similarly, Ami2 Di exhibits a higher structural similarity to the catalytic domain of N-acetylmuramoyl-L-alanine amidase from Paenibacillus polymyxa (PDB id: 1JWQ), with an RMSD of 1.84 Å calculated over 168 residues, compared to other amidases from various organisms.Similar to Ami1 Di , Ami2 Di exhibits a 65-residue disordered N-terminal region.Furthermore, a structural comparison of the top-ranked models for Ami1 Di and Ami2 Di , conducted using PyMOL, revealed a superimposed region shared by both proteins, potentially of significant functional relevance (Fig. S3).We identified the conserved active site motif -GHGG-in both amidases.In Ami1 Di , this motif is comprised of residues G160, H161, G162, and G163 (Fig. 5B), while in Ami2 Di , it is comprised of residues G396, H397, G398, and G399 (Fig. 5E).We have also identified the structurally equivalent residues for the zinc-binding sites in both Ami1 Di and Ami2 Di , as depicted in Fig. 5C and F. In Ami1 Di , these residues are H161, E175, H234, and N236, while in Ami2 Di , they are H397, E410, H468, and N470 (Fig. 5D and F).

Ami1 Di and Ami2 Di have peptidoglycan cleavage activity
Our experiments showed that both amidase Ami1 Di and Ami2 Di can perform PG splitting in E. coli cells.To investigate whether both D. indicus amidases can cleave D.
indicus cell wall, we purified C-terminus His-tag variant of both proteins (Fig. 6A).We observed that the purified Ami2 Di protein showed the presence of two bands in western blot (Fig S4B), one at full length of 65.6 kDa and a smaller band at 35 kDa (Fig. 6A).Heterologous expression of Deinococcus indicus proteins in E.coli could lead to protein stability or folding issues which may have caused partial degradation of Ami2 Di protein.Peptidoglycan cleavage activity was tested by dye release assay on RBB labeled PG.Purified Ami1 Di and Ami2 Di proteins Fig. 6 Peptidoglycan hydrolytic cleavage activity of Ami1 Di and Ami2 Di .(A) Western blot showing purified His-tagged Ami2 Di (65.6 kDa) -Lane 1 and Ami1 Di (42.5 kDa) -Lane 2 protein heterologously produced in E.coli BL21(DE3) strain (RP106 & RP105).M-Protein marker.(B) Determination of the PG hydrolase activity of Ami1 Di and Ami2 Di .Remazol brilliant blue (RBB)-labeled peptidoglycan was incubated with Ami1 Di (2 µM and 4µM) and Ami2 Di (2 µM and 4 µM), lysozyme as positive control and buffer as negative control at 37 °C.At 1 h and 12 h, the samples were pelleted down, and the absorbance of the supernatant was measured at 595 nm.The pictures below the panel show the results with an incubation of 12 h.Three biological replicate experiments were performed for each reaction, with error bars representing the standard deviation were incubated with RBB labeled PG for short (1 h) and long (12 h) -time intervals to measure peptidoglycan cleavage activity.At shorter incubation, only Ami2 Di showed significant enzymatic activity, corroborating our previous results that Ami2 Di may be a more potent PG hydrolase (Fig. 6B).Both enzymes showed increased cell wall hydrolysis at longer incubation period.Lysozyme was used as a positive control in the above experiment.At extended incubation times, PG degradation by both amidase enzymes was comparable to that obtained with lysozyme (Fig. 6B).Our results indicate that both Ami1 Di and Ami2 Di can act as PG hydrolase.

Amidase_3 domain is essential for the lytic activity of Ami1 Di
Both Ami1 Di and Ami2 Di proteins contain a C-terminal zinc-dependent catalytic domain known as the Ami-dase_3 domain (Fig. 1A).To investigate whether Ami-dase_3 domain is responsible for the hydrolytic activity of Ami1 Di , a C-terminus truncated version of Ami1 Di was generated.The C-terminus truncated variant of Ami1 Di (RP103) was unable to suppress the cell separation defects in E. coli amidase mutants (RP21), suggesting that the C-terminal catalytic domain is essential for PG cleavage (Fig. 7B).Histidine at the active site is crucial for both Zn 2+ binding and catalytic activity in amidases  [52,53].To investigate whether the active site residue plays a similar role in Ami1 Di , Histidine at 161 position was replaced with Alanine.The H161A active site variant (RP103) was unable to complement cell separation defects in E. coli amidase mutants (Fig. 7B).Quantitative analysis revealed only an 8% reduction in cell chains when the C-terminus truncated variant was used in the complementation assay, and this was further reduced to 2% when the active site H161A variant was expressed in E. coli ∆amiAC background (Fig. 7C).Taken together our data suggests that similar to other known amidases Histidine (His161) residue at the active site of Ami1 Di is essential for its catalytic activity.

Ami1
Di is a close structural homolog of AmiA Ec and displays a similar regulatory domain The crystal structure of E. coli AmiA has been recently studied [54] according to which both AmiA Ec and AmiB Ec contain regulatory domains that consist of a blocking helix (comprising residues 158-174 in AmiA Ec and 294-310 in AmiB Ec ) and an interaction helix (encompassing residues 180-192 in AmiA Ec and 320-332 in AmiB Ec ) [54].The blocking helix functions as an autoinhibitory regulator of AmiA, whereas the interaction helix serves as a binding site for EnvC [55].We assessed the structural similarity between Ami1 Di and Ami2 Di with AmiA (PDB id: 8C2O) and AmiB (PDB id: 8C0J) and computing the RMSD with the cealign command in PyMOL.Our analysis indicates that Ami1 Di exhibits a closer structural resemblance to AmiA, with an RMSD of 3.25 Å calculated over 216 residues.In contrast, Ami2 Di shows structural similarity to AmiB, with an RMSD of 2.82 Å calculated over 104 residues.Structural analysis was further extended to predict the regulatory domains and equivalent residues in Ami1 Di (Fig. 8A & B) compared to AmiA Ec .Our results predicted the presence of a regulatory domain in Ami1 Di comprising of a blocking helix (Arg259, Ser260, Leu261, Ala262, Val263, Arg264, Glu265, and Asn266) (Fig. 8C) and an interaction helix (Ser270, Leu271, Gly272, Glu273, Glu274, Leu275, Thr276, Arg277, Lys278, Ala279, Ala280, Ser281, Thr282, Ala283, Gln284, Asn285, Leu286, Leu287, and Gly288) (Fig. 8D).Our results indicate that Ami1 Di contains a regulatory domain, and its catalytic activity may be regulated comparably to E. coli amidases.However, the prediction of equivalent residues in the regulatory domain of Ami2 Di compared to AmiB Ec had lower coverage, primarily because of missing residues.
Fig. 8 Presence of regulatory domain in Ami1 Di (A) Schematic representation of the predicted regulatory domain of Ami1 Di comprising blocking helix and interaction helix.(B) Cartoon representation of Ami1 Di , colored based on secondary structure (Helices, sheets, and links are color-coded in red, yellow, and green, respectively).This representation is overlaid with AmiA Ec , displayed in gray.Notably, the blocking helix of AmiA Ec is highlighted in violet, while the interacting helix is shown in blue.(C) Highlight the corresponding blocking helix in Ami1 Di is shown in cyan, and the residues are labeled accordingly.The cartoon representation of AmiA is displayed in gray.(D) Highlight the corresponding interaction helix in Ami1 Di is shown in orange, and the residues are labeled accordingly.The Cartoon representation of AmiA Ec is shown in gray

Discussion
In this paper, we have characterized the amidases involved in peptidoglycan hydrolysis in D. indicus.D. indicus genome encodes two amidases, Ami1 Di and Ami2 Di .Both enzymes are able to supress cell separation defects in E. coli amidase mutants, indicating that Ami1 Di and Ami2 Di may play a role in septal peptidoglycan splitting.Most N-acetylmuramyl-l-alanine amidases fall into 3 families: NALAA-2, NALAA-3 and NALAA-5 [18].Members of the Amidase_3 family are zinc-dependent enzymes and include bacterial and phage amidases.Ami1 Di and Ami2 Di contain Amidase_3 catalytic domain at C-terminus, and comparative structural analysis identified the conserved active site motif -GHGG-in both amidases (Figs.1A and 5).In Ami1 Di , the active site residues comprised of G160, H161, G162, and G163 of these H161 is involved in zinc binding too (Fig. 5).Zn 2+ binding is essential for catalytic activity, and the H161A active site variant of Ami1 Di enzyme did not display PG splitting activity in E. coli (Fig. 7).The N-terminus is predicted to have signal peptide sequences (Fig. 1A).However, both proteins lack cell wall binding domains (CBD) and AMIN domain at N-terminus.The absence of CBD has also been observed in AmiC from C. crescentus [30] and M. tuberculosis amidase Rv3717 [56].
Preserving the integrity of the cell wall at all times is paramount for bacterial viability, and peptidoglycan cleavage activity of amidases is tightly controlled.In E. coli, amidase activation requires direct contact with LytM domain-containing protein EnvC and NlpD [11].NlpD specifically activates AmiC, while EnvC can activate both AmiA and AmiB.Recent studies revealed that activators NlpD and EnvC interact with their cognate amidases, displace the auto-inhibitory helix from the amidases' active site, and stimulate peptidoglycan hydrolase activity [23,25,54].The crystal structure of E. coli AmiA enzyme has a regulatory domain that consists of a blocking helix and an interaction helix [54].The blocking helix is involved in auto-inhibition and occludes the zinc active site, and the interaction helix mediates binding with activator EnvC [54].In our study, the AlphaFold2 structure of Ami1 Di displayed a high resemblance to E. coli AmiA.Ami1 Di is also predicted to have a regulatory domain consisting of both a blocking helix (259-266 residues) and an interaction helix (278-288 residues) (Fig. 8).This prompted us to predict that Ami1 Di may also be auto-inhibited and require interaction with an activator protein to stimulate PG cleavage.D. indicus genome mining revealed the presence of amidase activators LysM domain-containing peptidoglycan endopeptidase NlpC/ P60 (WP_088249103) and M23 family metallopeptidase EnvC (WP_229843994).EnvC Di has high sequence similarity with the E. coli homolog (31.4%) and also has the conserved metal-binding sites of the peptidase_M23 domain (Fig. S5).Taken together, our data suggest that the amidase/cognate circuit model may be operational in D. indicus.
AlphaFold2 structure of Ami2 Di protein revealed some significant differences compared to Ami1 Di .Ami2 Di was predicted to be a three-domain protein, comprising of a β-sandwich N-terminal domain, an α/β C-terminal domain, and an additional β-sandwich domain situated between the two primary domains (Fig. 5D).Moreover, overexpression of Ami2 Di in E. coli increased cell lysis, indicating that these enzymes may play functionally distinct roles in D. indicus.Both Deinococcus and Thermus genera are extremophiles known to survive harsh environmental conditions [57].The Deinococcaceae family has a unique cell envelope attributed to their survival in extreme environments.A representative member of this family, D. radiodurans, while stains gram-positive, has an envelope architecture of Gram-negative bacteria [58,59].D. radiodurans cell envelope consists of an inner membrane, a peptidoglycan layer, and an outer membrane [60][61][62][63].However, the outer membrane lacks classical lipopolysaccharides present in Gram-negative bacteria [64,65].Deinococcus also has a unique cell wall consisting of ornithine-Gly-peptidoglycan and differs from mDAP-peptidoglycan in E. coli [66,67].Both Ami1 Di and Ami2 Di could cleave D. indicus cell wall, indicating that both enzymes can hydrolyze ornithine-Gly-peptidoglycan.However, these enzymes may not be specific for ornithine-containing peptidoglycan as both enzymes were active in E. coli cells, too.Our study is the first to characterize cell wall amidases from Deinococcus indicus DR1 and future investigations would help to understand their role in the biogenesis and division of complex cell envelopes of these bacteria.

Fig. 1
Fig. 1 Domain architecture and phylogenetic analysis of D. indicus amidases.(A) Domain organization of N-acetyl-muramyl amidase of E. coli (AmiC), C. crescentus (AmiC), D. indicus Ami1 Di and Ami2 Di protein representing conserved Amidase_3 domain.(B) Phylogenetic trees.(i) Phylogenetic tree of Ami1 Di with the structural homologs having a Z-score of more than 10 from DALI.(ii) Phylogenetic tree of Ami2 Di with the structural homologs having a Z-score of more than 10 from DALI

Fig. 4
Fig.4 Overexpression of ami2 Di leads to cell lysis in E. coli.(A) Phase contrast images of strain RP101 and RP104 showing lytic activity of ami1 Di and ami2 Di , respectively on overexpressing under the control of L-arabinose promoter.Strain RP21, RP101 and RP104 were grown till OD 600 ∼ 0.2 and induced with L-arabinose for 10 h.Cells were collected, and about 6 µL sample was immobilized on 1XPBS agarose pad and imaged.Cells overexpressing ami2 Di (RP104) show higher lytic activity as represented by dead cells.(B) Growth curve assay confirming the elevated lytic activity of ami2 Di (RP104).Strain RP21, RP101, and RP104 were induced with L-arabinose at OD 600 ∼ 0.1.Optical density was measured every 1 h.Cells induced with 0.2% glucose were used as the negative control.Datasets are from three biological replicates, and error bars represent standard deviation

Fig. 5
Fig. 5 Structural analysis of Ami1 Di and Ami2 Di showing zinc binding and active site motifs.(A) Cartoon representation of Ami1 Di , colored based on secondary structure (helix, sheet, and loop are shown in red, yellow, and green, respectively).(B) Close-up on the conserved active site motif of Ami1 Di .The motifs are represented as sticks, where nitrogen and oxygen are shown in blue and red, respectively.(C) Close-up on the equivalent residue for zinc binding sites of Ami1 Di .The zinc and surrounding helix and sheets are shown in green and bright orange, respectively.The chelating residues are represented as sticks (nitrogen and oxygen are shown in blue and red, respectively).(D) Cartoon representation of Ami2 Di , colored based on secondary structure (helix, sheet, and loop are shown in red, yellow, and green, respectively).(E) Close-up on the conserved active site motif of Ami2 Di .The motifs are represented as sticks, where nitrogen and oxygen are shown in blue and red, respectively.(F) Close-up on the equivalent residue for zinc binding sites of Ami2 Di .The zinc and surrounding helix and sheets are shown in green and bright orange, respectively.The chelating residues are represented as sticks (nitrogen and oxygen are shown in blue and red, respectively)

Table 1
Strains and plasmids used in this study ECL Western Blotting Substrates (Bio-Rad Laboratories, Inc. U.S.A) according to the manufacturer's protocols.